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1 Structural basis of specific trna aminoacylation by a small in vitro selected ribozyme Hong Xiao 1, Hiroshi Murakami 2, Hiroaki Suga 2, 3, & Adrian R. Ferré-D'Amaré 1 * 1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle WA , USA 2 Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan 3 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, , Tokyo, Japan * To whom correspondence should be addressed, aferre@fhcrc.org; phone (206) ; facsimile (206)

2 Supplementary Figure 1. Predicted (prior to structure determination) secondary structure of flexizyme 1, color coded as in Fig. 1. Non-essential RNA segments are shown as thick lines. Secondary structure of RNA segments named in black type (P1, P2, P3) was predicted based on covariation and biochemical analyses. Predicted base pairs between the 3'-terminal residues of flexizyme and positions t73-t75 of the substrate RNA (trna, a minihelix, or a microhelix) are indicated by the dashed lines. RNA segments in gray type (P1a, J1a/2, J2/1a, J1a/3) are named according to the results of crystallographic analysis. ASD indicates that in the crystal structure these two segments pair, forming the irregular active site duplex. Note that the base-pair register of P1 also differs between the prediction and the crystal structure (compare with Fig. 1a). 2

3 Supplementary Figure 2. Model of felxizyme interacting with full-length yeast trna Phe. The acceptor and T stems of the trna (Supplementary ref. 2) were superimposed on the minihelix of our structure using the least-squares superposition function of program O (Supplementary ref. 3). This shows that trna can be accommodated on flexizyme in a manner analogous to the observed mode of interaction with the minihelix RNA without any steric clashes. The closest approach between loop L1 (dark blue) of flexizyme and the base of the D-stem of trna Phe is ~5 Å. 3

4 Supplementary Figure 3. Portion of the final residual F o F c Fourier synthesis calculated with phases from the refined crystallographic model without inclusion of the phenylalanine and amplitudes from Crystal III, contoured at 2.5 (blue mesh) and 3.5 (red mesh) standard deviations above mean peak height (s.d.) To facilitate comparison with other figures (all made with the model resulting from refinement against Crystal II data) this Crystal III map is superimposed on the crystallographic model that resulted from the refinement carried out against the 2.8Å amplitudes from Crystal II. The large feature in the middle of the figure was assigned to the benzyl group of L-phenylalanine in the most common rotamer of the amino acid. This places the reactive carbonyl in Van der Waals contact (3.6 Å) of the 3'-OH of the trna minihelix. The orientation of the carboxylic acid of the modeled phenylalanine is compatible with the leaving group to point away from the active site, and with its π-face orthogonal the nucleophile, as expected for the reaction 4. Electron density that corresponds to three water molecules, one of which is a water of hydration of the bound magnesium ion, and another packing on the phenyl ring of the amino acid 5, is also apparent. At the current resolution, only two of the hydrationdoi: /nature

5 shell waters of the cation are visible in residual F o - F c Fourier syntheses, but the magnesium RNA distances imply that its octahedral hydration shell is intact. 5

6 Supplementary Figure 4. Shape complementarity or solvent accessible area burial are not the primary determinants of amino acid discrimination by flexizyme. The side chain of the L-phenylalanine bound to the ribozyme was replaced with a common rotamer of the corresponding amino acid that would not clash with the RNA (keeping the backbone atoms in the same position). Shape complementarity of the various amino acids and the active site surface they abut (determined using program SC (Supplementary ref. 6) with a probe radius of 1.4Å and the default trim of 1.5Å), solvent accessible area buried, and relative specificity of flexizyme 1 is as follows: (L-phenylalanine, 0.68, Å 2, 1.0); (D-phenylalanine 0.68, Å 2, 0.18); (Lmethionine, 0.72, Å 2, 0.001); (L-glycine, 0.7, 46.7 Å 2, ); (L-leucine, 0.67, 96.8 Å 2, ); (L-valine, 0.71, 80.5, ); (L-glutamine, 0.76, Å 2, <0.0001). Specificity correlates poorly with shape complementarity or accessible surface area buried. In contrast, flexizyme displays a clear preference for aromatic amino acids. Note (panel A) how D- 6

7 phenylalanine can pack against the side of the active-site pocket opposite from where L- phenylalanine binds. This side of the pocket is shallower than the L-phenylalanine binding pocket (presumably accounting for the ~ 5-fold lower activity of the D- isomer), but presents a planar surface for the D-phenylalanine to pack against. The preference of flexizyme for phenylalanine is also interesting in that PheRS is a class II ARS (even though, uniquely among enzymes of this class, it initially charges the 2'-OH). 7

8 Supplementary Figure 5. Comparison of the sequences of flexizyme and dinitro-flexizyme, a variant that recognizes the dinitrobenzyl esters of amino acids. A, sequence and secondary structure of flexizyme. B, sequence of dinitroflexizyme 7, arranged to highlight similarities (same color-coding as flexizyme) and differences (green) with flexizyme. C, Phe-CME, the activated L-phenylalanine derivative, substrate for flexizyme. D, Dinitrobenzyl ester (R = amino acid side-chain) substrate for dinitro-flexizyme. In addition to the structural similarity of the ribozymes, note that both RNAs employ aromatic substrates. See Supplementary refs 7 and 8 for details. 8

9 Supplementary Figure 6. "Closed" conformation of the active site of molecule B of flexizyme. A, B Comparison of the B-factors of the active sites of molecules A (left) and B. The B-factors range from ~60 Å 2 (blue), ~80 Å 2 (green), ~130 Å 2 (yellow), to ~160 Å 2 (red). C Electron density (σa-weighted 2 F o - F c synthesis calculated with the final model and amplitudes from Crystal II, contoured at 1.25 s.d.) in the active site of molecule B. Despite the higher B-factors, the position of the nucleotides of the pocket, and of the terminal ta76 of the minihelix are well 9

10 defined. Note how the unpaired U47 has buckled into the phenylalanine binding pocket (compare with Fig. 3A). 10

11 Supplementary Figure 7. Evidence for dephosphorylation of the 3'-terminus of the crystallization construct. A, Optimization of reaction conditions employing a 24 nt model minihelix RNA bearing a 5'-triphosphate and a 2',3' cyclic phosphate (lane 1). The minihelix RNA was generated by in vitro transcription with phage T7 RNA polymerase followed by cleavage using the VS ribozyme. The minihelix was treated with 10 mm HCl at room temperature for 30' to open (but not remove) the cyclic phosphate 9. This results (lane 2) in slightly faster mobility than the input RNA (after taking into account the 'smiling' of the gel). Treatment of the minihelix with T4 polynucleotide kinase (PNK) under the conditions described 11

12 in Methods, but with the ATP omitted, results in cyclic phosphate opening and partial removal. The RNA with the opened cyclic phosphate comigrates with the RNA with the acid-opened cyclic phosphate in lane 2, and the dephosphorylated form migrates more slowly than the input RNA (lane 3). ATP is known to stabilize PNK (Supplementary ref. 10). Treatment of the oligonucleotide under the conditions described in Methods (including ATP), results in complete opening and removal of the cyclic phosphate (lane 4). RNAs (2 µg in each lane) were resolved on a 8M urea, 20% polyacrylamide gel, and visualized by staining with toluidine blue. Under the conditions employed, toluidine blue staining can detect at least 50 ng/band of RNA. Thus, it appears that PNK treatment of the minihelix, in the presence of 1 mm ATP, results in at least 97.5% opening and removal of the cyclic phosphate. B, Detail of a composite simulatedannealing omit 2 F o - F c electron density map 11 corresponding to molecule A from Crystal II, contoured at one s.d. around the 3' terminus of the crystallization construct (gray mesh), shows no evidence of a cyclic phosphate on the terminal ribose (red furanose ring). Simulatedannealing omit electron density is shown together with the residual F o - F c electron density (from Crystal III refinement) and final model as shown in Supplementary Figure

13 Supplementary Figure 8. Unbiased electron density. Portion of the 2.8 Å resolution unbiased electron density map resulting from phase combination of MAD phases and PHASER molecular replacement phases (Crystal I and II data), contoured around the 5'-triphosphate of molecule A, superimposed on the final crystallographic model (1.2 s.d.) The model used to generate the molecular replacement phases did not include residues G1, G51, the 5'-triphosphate, the innersphere coordinated magnesium ions (magenta spheres) or the coordination water (red sphere). These two inner-sphere coordinated magnesium ions may correspond to those detected biochemically 12. The 5'-triphosphate and its two coordinated magnesium ions are unusually well-ordered, and lie in the major groove of helix P1. The 2'-OH of residue G51 from the J1a/3 hairpin turn abuts the 5'-oxygen of G

14 Supplementary Table 1 Crystallographic Statistics Crystal I, Inflection Crystal I, Peak Crystal II Crystal III Data collection Space group C2 C2 C2 Cell dimensions a, b, c (Å), α, β,γ ( ) , 48.05, 91.07, 90, 93.96, , 48.73, 90.52, 90, 93.51, ,48.08, 90.65, 90, 93.50, 90 Wavelength (Å) Resolution (Å) a ( ) ( ) ( ) ( ) R merge (%) a 5.6 (48.4) 5.8 (54.1) 4.7 (37.2) 8.7 (50.1) <I>/<σ(I)> a 22.1 (3.1) 25.4 (2.8) 28.8 (3.3) 21.1 (1.9) Completeness (%) a 99.7 (99.9) 99.8 (100.0) 99.1 (99.7) 80.8 (51.6) Redundancy a 4.6 (4.7) 6.0 (5.9) 3.6 (3.5) 6.4 (2.9) Phasing Phasing power Iso (acentrics) n/a Ano (acentrics) R Cullis (%) Mean overall figure of merit Refinement Resolution (Å) a ( ) ( ) No. reflections b (4011) (1355) R work/r free (%) 27.9/ /24.7 No. atoms RNA Protein Ion Water 10 0 Mean B-factors (Å 2 ) RNA Protein Ion Water 65.3 n/a R.m.s. deviations Bond lengths (Å) Bond angles ( ) Luzzati coordinate precision (Å) b 0.47 (0.51) 0.46 (0.55) PDB accession code 3CUL 3CUN a Values in parentheses are for the highest resolution shell. b Values in parentheses are for the cross-validation set. One crystal was employed for each data-set. 14

15 Supplementary References 1. Saito, H., Watanabe, K. & Suga, H. Concurrent molecular recognition of the amino acid and trna by a ribozyme. RNA 7, (2001). 2. Shi, H. & Moore, P. B. The crystal structure of yeast phenylalanine trna at 1.93 Å resolution: a classic structure revisited. RNA 6, (2000). 3. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, (1991). 4. Piccirilli, J. A., McConnell, T. S., Zaug, A. J., Noller, H. F. & Cech, T. R. Aminoacyl esterase activity of the Tetrahymena ribozyme. Science 256, (1992). 5. Dougherty, D. A. Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science 271, (1996). 6. Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, (1993). 7. Murakami, H., Ohta, A., Ashiai, H. & Suga, H. A highly flexible trna acylation method for non-natural polypeptide synthesis. Nature Methods 3, (2006). 8. Ohta, A., Murakami, H., Higashimura, E. & Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem Biol 14, (2007). 9. Price, S. R., Ito, N., Oubridge, C., Avis, J. M. & Nagai, K. Crystallization of RNAprotein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J. Mol. Biol. 249, (1995). 15

16 10. Galburt, E., Pelletier, J., Wilson, G. & Stoddard, B. Structure of a trna Repair Enzyme and Molecular Biology Workhorse. T4 Polynucleotide Kinase. Structure (Camb) 10, (2002). 11. Brünger, A. T. et al. Crystallography and NMR system: a new software system for macromolecular structure determination. Acta Crystallogr. D54, (1998). 12. Saito, H. & Suga, H. Outersphere and innersphere coordinated metal ions in an aminoacyl-trna synthetase ribozyme. Nucleic Acids Res 30, (2002). 16